This invention relates to bioreactors which can be used to enable cells to produce cell products, and particularly to enable hybridomas to produce monoclonal antibodies.
The bioreactor system resembles a living organism in many ways. It has an enclosure in which cells can live, multiply, and produce; a circulating system for supplying water and nutrient and removing waste from the cells; a respiratory system for providing oxygen and removing carbon dioxide; and means for removing cell products and wastes from the system. The success of the whole system depends not only on how well each of the elements is designed but even more on how well they interact and coordinate with each other to maintain effective conditions for the production of cell products.
Advances in biomedical technologies have initiated a demand for mammalian cell products. The diagnostic, prophylactic, and therapeutic applications for such cell products appear vast and an ever-expanding variety of possibilities will generate new markets far into the future. The exploding demand for mammalian cell products has spurred considerable research into mass cell culture techniques and bioreactors.
Currently, several bioreactor designs have been developed, used and marketed by adapting existing devices for use in the realm of mammalian cell culture. The two technologies most commonly exploited in this fashion are those of bacterial fermenters (batch culture), and hollow fiber dialysis units modified for stationary cell culture. Each system brings with it certain inherent advantages, but there are also certain limitations and disadvantages with regard to mammalian cell culture.
Bulk fermentation in batch operations is limited by the absence or great difficulty of the removal of cell products, and particularly toxic materials and other materials which inhibit cell metabolism or the production of certain cellular products. In addition, conditions of bulk fermentation are generally based on air or oxygen spargers to provide for the oxygen requirements of the cells, which makes for excessively turbulent conditions within the reactor. Sterility in large fermentation tanks is a difficult problem, and when contamination occurs, the losses in considerable labor and production time and the expensive materials when the batch is dumped cn be quite large.
Various considerations have made immobilized cell bioreactors preferable over alternative systems for the commercial production of monoclonal antibodies as well a other mammalian cell products. Immobilized hybridoma cells in particular can produce their products virtually indefinitely in such bioreactors without disturbances related to the harvesting of products. However, adequate oxygenation of the cultured cells and removal of carbon dioxide has been a limiting factor in the development of efficient and economical designs.
Most immobilized-cell bioreactor designs in the prior art deliver oxygen dissolved in the nutrient medium to the cells and remove carbon dioxide as it dissolves in the same medium. Since oxygen is rapidly depleted, large volumes of nutrient medium must be circulated through the bioreactor to meet cellular needs. The flow rates based on oxygen consumption are generally adequate for waste removal. The large volumes of circulating nutrient medium necessary for scaled-up producton bioreactors of this kind require large pumps which are expensive and greatly increase the size of the bioreactor unit. High nutrient medium flow rates also increase fluid pressure and turbulence in the system, with the concurrent risk of substantial losses of both production cells and of relatively expensive nutrient components through the physical stresses imposed, particularly high shear conditions.
A diverse variety of approaches have been employed in the development of membrane based cellular bioreactors. See for example the varying approaches illustrated in U.S. Pat. Nos. 3,997,396, 4,087,327, 4,201,845, and 4,537,860.
In 3,997,396, attachment dependent cells are attached on the outer surface of hollow fiber membranes, and derive their oxygen supply from an air or other oxygen carrier flowing on the inside of the fiber. In this approach, the number of cells is limited to a monolayer on the available membrane surface, limiting the cell density to low levels. In addition, with pure oxygen, most cells are limited in their ability to survive the high oxygen tensions. If the applied gas pressure is too high, it may produce gas bubbles that will disrupt the cells or their attachment. On the other hand, the requirement that the cells attach to the surfaces requires that the membrane be hydrophilic, and thus susceptible to wetting by the nutrient, and passage of materials into and even through the pores, causing clogging and disruption of the flow. This will dictate a high gas pressure to limit the rate of these phenomena, and damage to the cells will inevitably result.
A different approach is taught in 4,087,327; in this patent it appears that the outer surfaces of hollow fiber membranes or solid fibers are used as attachment sites for attachment dependent cells, but the interior of the fibers, when hollow fibers are employed, does not appear to be used. Oxygen is apparently transported to the cells dissolved in the nutrient. The nutrient is pumped by pressure transversely through the cell chamber, subjecting the cells to undesirable pressure and flow turbulence which can damage the cells.
U.S. Pat. No. 4,201,845 is a Continuation-in-Part of 4,087,327, supra. In this version, the interior of hollow fiber membranes are used for oxygen transport into the cell reaction zone. The nutrient is again pumped transversely through the cell chamber, with the attendant problems. The difficulties with this teaching are the same as those noted above with respect to 3,997,396.
U.S. Pat. No. 4,537,860 teaches a cell reactor which employs an annular construction of porous cylinders of polymer or ceramic materials to confine cells, pump nutrient through the cell chamber (with all the attendant difficulties of through flow operations), and semipermeable, silicone rubber tubing to carry gas into, and to cause it to perfuse into, the cell chamber. The distance of the cells can be as much as 1 to 2 cm from the oxygen supply tube, and coupled with the properties of the silicone rubber tubing, leads to the requirement that the oxygen source be fed under such pressure that the supply tube passes gaseous phase into the reaction zone in order to provide adequately oxygen to the cell chamber, with attendant turbulence, high internal pressures and damage to the cells. This "sparger" approach leads to inadequate rates of oxygen uptake to assure cell producivity and even viability in substantial parts of the vessel.